Project Summary Tuberculosis (TB) is the leading cause of death from bacterial infection. During infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress in the host cells, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and chemotherapy. These stresses impair protein integrity and result in protein aggregation. The impaired or aggregated proteins have to be either degraded and recycled or else resolved and repaired. Protein degradation and recycling in Mtb is mainly carried out by the proteasome system, while protein rescue (i.e., resolving toxic protein aggregates to a native folded state) is carried out by the ATP-powered ClpB/DnaK bi-chaperone system. Therefore, the proteasome and the ClpB/DnaK systems are the yin and yang of cellular proteostasis in Mtb. Interestingly, the proteasome is absent in most bacteria; it is present only in the order of Actinomycetales and is essential to Mtb's survival inside the host. Genetic studies have established the Mtb proteasome as an ideal target for the development of anti-TB agents. In collaboration with chemical biology labs, we propose to study the binding poses of several anti-TB agents that are metabolically stable and that specifically inhibit the Mtb 20S proteasome core particle while sparing the human constitutive and immunoproteasome (Aim 1). In the previous funding cycle, we solved the crystal structures of the ATP-dependent proteasomal activator, the Mpa hexamer, as well as the ATP-independent proteasomal activator called the PafE dodecamer. We will next investigate how partially unfolded or oxidized protein substrates are recognized and targeted for degradation by the Mtb proteasome (Aim 2). It is notable the transcriptional repressor HspR of the Mtb ClpB/DnaK bi-chaperone system is a substrate of the Mtb PafE- proteasome system; hence, the Mtb proteasome directly regulates the bi-chaperone system. We plan to study the structure and function of the ATP-driven disaggregation machinery, i.e., the Mtb ClpB hexamer (Aim 3), as part of the long-term plan to characterize the complete ClpB/DnaK bi-chaperone system. Characterizing the two opposing systems of protein degradation and protein reactivation lays the groundwork for the development of a combination approach that simultaneously targets both systems, leading to irreparable disruption of the microbial proteostasis and synergistic killing of nonreplicating bacteria. Overall, the proposed work will improve our understanding of the molecular mechanism of protein homeostasis in Mtb, the deadliest bacterial pathogen.